1. Technical Field
The present invention is concerned with methods to determine particle size by image analysis, particularly by scanning electron photomicrographs.
2. Background Art
There are an ever-increasing number of organic compounds being formulated for therapeutic or diagnostic effects that are poorly soluble or insoluble in aqueous solutions. Such drugs provide challenges to delivering them by the administrative routes detailed above. Compounds that are insoluble in water can have significant benefits when formulated as a stable suspension of sub-micron particles. Accurate control of particle size is essential for safe and efficacious use of these formulations. Particles must be less than seven microns in diameter to safely pass through capillaries without causing emboli (Allen et al., 1987; Davis and Taube, 1978; Schroeder et al., 1978; Yokel et al., 1981). One solution to this problem is the production of small particles of the insoluble drug candidate and the creation of a microparticulate or nanoparticulate suspension. In this way, drugs that were previously unable to be formulated in an aqueous based system can be made suitable for intravenous administration. Suitability for intravenous administration includes small particle size (<7 μm), low toxicity (as from toxic formulation components or residual solvents), and bioavailability of the drug particles after administration.
Preparations of small particles of water insoluble drugs may also be suitable for oral, pulmonary, topical, ophthalmic, nasal, buccal, rectal, vaginal, transdermal administration, or other routes of administration. The small size of the particles improves the dissolution rate of the drug, and hence improving its bioavailability and potentially its toxicity profiles. When administered by these routes, it may be desirable to have particle size in the range of 5 to 100 μm, depending on the route of administration, formulation, solubility, and bioavailability of the drug. For example, for intravenous administration, it is desirable to have a particle size of less than about 7 μm. For pulmonary administration, the particles are preferably less than about 10 μm in size.
Determination of the size of these particles has been routinely performed by image analysis using optical light microscopy. However, performing accurate and precise analysis of small particles, especially those below one micron, becomes difficult due to the limited number of pixels available for each particle and also the practical (and theoretical) limits of resolution of the light microscope. Moreover, image analysis of particles approximately less than or equal to 1 μm in suspension is not feasible due to the Brownian motion of the particles and their constant motion in X, Y and Z directions on a standard slide preparation.
Image analysis of particles can be performed by a variety of methods and algorithms. Typically, a particle is positioned onto a depth filter and the image of the particle is isolated using a threshold algorithm to separate the image of the particle from the image of the background. In the case of particles less than 1 μm, a variety of problems occur. For example, depth filters do not present all the particles (some are embedded into the filter matrix) nor do they present all the particles normal to the optical axis. While nuclear track etched membrane filters present a smooth, flat surface perforated by more or less circular holes, these membranes do not provide for sufficient contrast between the particles and the membrane surface. Additionally, the holes of the nuclear track etched membrane filter may be detected as particles, thus causing another source of error.
The present invention discloses a method to determine particle size using image analysis. The method is particularly suitable for small particles such as those that are less than 1 μm in size.